Treatment of diseases related to igfb3 and its receptor

ABSTRACT

Insulin-like growth factor-binding protein 3 receptor (IGFBP-3R) agonists and methods of their use to treat diseases involving IGFBP-3 and IGFBP-3R are provided. The agonists may be antibodies or other molecules specific for binding to and activating IGFBP-3R. The agonists are used to treat e.g. cancer, metabolic syndrome and obstructive respiratory disorders. In addition, methods of diagnosing cancer and predicting the chance of recurrence, metastasis and/or survival by measuring the level of IGFBP-3R in tumor tissue are provided.

BACKGROUND OF THE INVENTION Field of the Invention

The invention generally relates to methods of treating diseasesinvolving insulin-like growth factor-binding protein 3 (IGFBP-3) and itsreceptor, IGFBP-3R. In particular, the invention provides methods ofdiagnosing cancer, and treating cancer, metabolic syndrome andobstructive respiratory disorders using IGFBP-3R agonists.

Background of the Invention

The American Cancer Society estimates that almost 1.7 million new casesof cancer will be diagnosed in 2017. With respect to cancer mortality,lung cancer is by far the leading cause of cancer death among females(25%), followed by breast (14%), and colorectal (8%) cancers. Amongmales, lung (27%), colorectal (9%) and prostate (8%) cancers are theleading causes of cancer death. New therapy for those high mortalitycancers is urgently needed. Oncology drug discovery currently suffersfrom a very acute paucity of selective and drug-accessible moleculartargets in which to attack tumor cells.

In particular, according to the American Cancer Society, about 1 in 8(12%) women in the US will develop invasive breast cancer (BC) duringtheir lifetime and about 40,000 women will die from BC in 2016. Inparticular, triple negative breast cancer (TNBC), constituting aheterogeneous subtype of BC that lacks expression of the drug targetsER, PR and HER2, accounts for 15-20% of all diagnosed BC cases, and yetis responsible for a disproportionate number of cancer-related deaths¹.TNBC does not respond to hormonal therapy (such as tamoxifen oraromatase inhibitors) or therapies that target HER2 receptors, such asHerceptin (trastuzumab). Current treatment mainly relies on chemo- andradiation-therapy as there are no targeted therapies specificallyapproved for TNBC. Despite initial responses to chemotherapy andradiation-therapy, resistance frequently and rapidly develops. Given thecomplexity of TNBC biology and the lack of “traditional” therapeutictargets, new targeted approaches are, therefore, urgently needed.

Metabolic syndrome is a serious health condition that is becoming moreand more prevalent as frequency of obesity and sedentary lifestylesarise, and as a result of aging populations. For example, in the UnitedStates, about 34% of the population has metabolic syndrome, and theprevalence increases with age: metabolic syndrome affects about 60% ofthe U.S. population older than age 50. Metabolic syndrome is associatedwith an increased risk of several debilitating diseases, includinginsulin resistance, atherosclerotic cardiovascular disease (e.g., heartdisease and stroke) and type 2 diabetes. The development of thesediseases results in a high negative impact on the quality of life ofthose who are afflicted, and places a high burden on the alreadystrained health care systems of countries. While some treatments areavailable for specific symptoms (e.g. drugs for high blood pressure,etc.), and while life style changes can have a positive impact, allpatients do not respond equally well to medications or to the need forlife style changes. It would be beneficial to have available additionalmedicaments to treat metabolic syndrome.

Obstructive respiratory disorders (also known as obstructive lung orpulmonary disease) is a category of respiratory disease characterized byairway obstruction. Several diseases are included in this category,including chronic obstructive pulmonary disease (COPD) and asthma. Theincidence of these maladies is on the rise. For example, according toestimates from the Global Burden of Disease Study, COPD, which afflictsboth smokers and non-smokers, afflicted more than 300 million peopleworldwide in 2013, with 250,000 annual deaths attributed to the disease.The disease burden and its financial impact is predicted to increase,e.g. due to population aging. As of 2014, it was estimated that asthmaaffected as many as 334 million people worldwide. It is the most commonchronic disease in children and its prevalence is also rising. Whilethere are some medications available to control symptoms, there is anongoing need to provide additional improved methods and agents fortreating these and other types of obstructive respiratory disorders.

SUMMARY OF THE INVENTION

Various features and advantages of the present invention are set forthin the description of invention that follows, and in part will beapparent from the description or may be learned by practice of theinvention. The invention will be realized and attained by thecompositions and methods particularly pointed out in the writtendescription and claims hereof.

This disclosure describes a critical antitumor, anti-inflammatorysignaling cascade, the IGFBP-3/IGFBP-3R axis. It has been discoveredthat IGFBP-3 and its receptor, IGFBP-3R play a role in several diseases,including cancer, metabolic syndrome, obstructive respiratory disordersand various inflammatory disorders. (IGFBP-3R is also known as“transmembrane protein 219” and “IGFBP-3R” and the acronym “TMEM219” maybe used interchangeably herein.) For such diseases, in some aspects, ithas been determined that the level of IGFBP-3 that is produced isinsufficient to cause sufficient activation of IGFBP-3R (TMEM219).Therefore, the present disclosure provides agents that substitute forthe natural ligand IGFBP-3. The agents are TMEM219 agonists which bindto and activate TMEM219 and can thus be used to prevent, treat orameliorate symptoms of such diseases and/or in some cases, therecurrence of the diseases, and/or improve the prognosis (e.g. survivalrate, rate of relapse, disease free survival time, etc.) of patientssuffering from the diseases. In one aspect, the TMEM219 agonists aremonoclonal antibodies (mAbs). Thus, this disclosure describes thetherapeutic use of TMEM219 agonist mAbs to treat cancer (includingbreast, colon and lung cancer), metabolic syndrome, obstructiverespiratory disorders, inflammatory disorders, and related diseases. Inparticular, TMEM219 agonistic antibodies constitute a new generation oftherapeutics with a unique mechanism and target specificity for treatingthese disorders. The TMEM219 agonist mAbs advantageously exhibit nodeleterious harmful effects (such as cell damage or cell killing) onnormal, non-disease (e.g. non-tumor) cells.

In addition, with respect to cancer diagnosis, it has been discoveredthat a low level of expression of TMEM219 in tumor cells is indicativeof a poor prognosis, e.g. an increased risk of metastasis, recurrenceand/or a lower overall chance of survival. Thus, for patients withlevels of expression of TMEM219 that are lower than a predetermined,corresponding reference value, an aggressive treatment regimen istypically recommended. Conversely, patients with a high level ofexpression of TMEM219 in tumor cells have a relatively good prognosis,with a lower risk of metastasis and recurrence and/or a higher chance ofsurvival. Accordingly, a less aggressive (and thus less toxic) treatmentregimen with fewer side effects is recommended.

It is an object of this disclosure to provide an agonist that binds toand activates TMEM219. In some aspects, the agonist is a small molecule,a peptide, a polypeptide or an antibody. In other aspects, the agonistis an antibody containing at least one complementarity determiningregion (CDR) selected from the group consisting of: SEQ ID NO: 15, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, the amino acidsequence ATS, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, the amino acid sequence SAS, and SEQ ID NO: 33, or a CDR that is atleast 90% identical to the at least one CDR. In further aspects, theantibody comprises i) a heavy chain with an amino acid sequence that isat least 90% identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5,and/or ii) a light chain with an amino acid sequence that is at least90% identical to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. Inadditional aspects, i) the heavy chain is at least 90% identical to SEQID NO: 1 and the light chain is at least 90% identical to SEQ ID NO: 2;ii) the heavy chain is at least 90% identical to SEQ ID NO: 3 and thelight chain is at least 90% identical to SEQ ID NO: 4; or iii) the heavychain is at least 90% identical to SEQ ID NO: 5 and the light chain isat least 90% identical to SEQ ID NO: 6. In some aspects, the antibodycomprises a detectable label.

The disclosure also provides a method of treating an TMEM219 expressingcancer in a patient in need thereof, comprising: administering to saidpatient a therapeutically effective amount of an agonist that binds toand activates TMEM219. In some aspects, the agonist is a small molecule,a peptide, a polypeptide or an antibody. In other aspects, the agonistis an antibody containing at least one complementarity determiningregion (CDR) selected from the group consisting of: SEQ ID NO: 15, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, the amino acidsequence ATS, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, the amino acid sequence SAS, and SEQ ID NO: 33, or a CDR that is atleast 90% identical to the at least one CDR. In further aspects, theantibody comprises i) a heavy chain with an amino acid sequence that isat least 90% identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5,and/or ii) a light chain with an amino acid sequence that is at least90% identical to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. Inadditional aspects, i) the heavy chain is at least 90% identical to SEQID NO: 1 and the light chain is at least 90% identical to SEQ ID NO: 2;ii) the heavy chain is at least 90% identical to SEQ ID NO: 3 and thelight chain is at least 90% identical to SEQ ID NO: 4; or iii) the heavychain is at least 90% identical to SEQ ID NO: 5 and the light chain isat least 90% identical to SEQ ID NO: 6. In some aspects, the cancer isbreast cancer, colon cancer, lung cancer, ovarian cancer, pancreaticcancer, liver cancer or leukemia.

The disclosure also provides a method of treating insulin resistance ina patient in need thereof, comprising: administering to said patient atherapeutically effective amount of an agonist that binds to andactivates TMEM219. In some aspects, the agonist is a small molecule, apeptide, a polypeptide or an antibody. In other aspects, the agonist isan antibody containing at least one complementarity determining region(CDR) selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, the amino acid sequenceATS, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, theamino acid sequence SAS, and SEQ ID NO: 33, or a CDR that is at least90% identical to the at least one CDR. In further aspects, the antibodycomprises i) a heavy chain with an amino acid sequence that is at least90% identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, and/or ii)a light chain with an amino acid sequence that is at least 90% identicalto SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. In additional aspects, i)the heavy chain is at least 90% identical to SEQ ID NO: 1 and the lightchain is at least 90% identical to SEQ ID NO: 2; ii) the heavy chain isat least 90% identical to SEQ ID NO: 3 and the light chain is at least90% identical to SEQ ID NO: 4; or iii) the heavy chain is at least 90%identical to SEQ ID NO: 5 and the light chain is at least 90% identicalto SEQ ID NO: 6.

The disclosure further provides a method of treating an inflammatorydisorder in a patient in need thereof, comprising: administering to saidpatient a therapeutically effective amount of an agonist that binds toand activates TMEM219. In some aspects, the agonist is a small molecule,a peptide, a polypeptide or an antibody. In other aspects, the agonistis an antibody containing at least one complementarity determiningregion (CDR) selected from the group consisting of: SEQ ID NO: 15, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, the amino acidsequence ATS, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, the amino acid sequence SAS, and SEQ ID NO: 33, or a CDR that is atleast 90% identical to the at least one CDR. In further aspects, theantibody comprises i) a heavy chain with an amino acid sequence that isat least 90% identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5,and/or ii) a light chain with an amino acid sequence that is at least90% identical to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. Inadditional aspects, i) the heavy chain is at least 90% identical to SEQID NO: 1 and the light chain is at least 90% identical to SEQ ID NO: 2;ii) the heavy chain is at least 90% identical to SEQ ID NO: 3 and thelight chain is at least 90% identical to SEQ ID NO: 4; or iii) the heavychain is at least 90% identical to SEQ ID NO: 5 and the light chain isat least 90% identical to SEQ ID NO: 6. In some aspects, the antibodycomprises a detectable label. In some aspects, the inflammatory disorderis selected from the group consisting of ulcerative colitis, colitis,Crohn's disease, atherosclerosis, chronic peptic ulcer, chronicobstructive lung disease, idiopathic pulmonary fibrosis, tuberculosis,arthritis, chronic sinusitis, asthma, hepatitis, ankylosing spondylitis,liver fibrosis, non-alcoholic steatohepatisis or chronic periodontitis.In additional aspects, the arthritis is osteoarthritis, rheumatoidarthritis (RA), and psoriatic arthritis.

The disclosure also provides a method of determining a prognosis of asubject with cancer and treating the subject accordingly, comprising, i)measuring a level of TMEM219 expression in a tumor sample from thesubject ii) comparing the level of TMEM219 expression obtained in stepi) with a corresponding reference level of TMEM219 expression; and iii)if the level of TMEM219 expression is the same or lower than thecorresponding reference level of TMEM219 expression, then iv) concludingthat the patient has a poor prognosis and providing an aggressiveanti-cancer treatment to the patient; Or v) if the level of TMEM219expression is higher than the corresponding reference level of TMEM219expression, then iv) concluding that the patient has a good prognosisand providing a less aggressive anti-cancer treatment to the patient. Insome aspects, the cancer is breast cancer, colon cancer, lung cancer,ovarian cancer, pancreatic cancer, liver cancer or leukemia. Inadditional aspects, the prognosis includes one or more of risk ofrecurrence of the cancer in the patient, risk of metastasis, overallsurvival of the patient and prediction of the benefit of chemotherapyfor the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B. IGFBP3 and TMEM219 (IGFBP-3R) gene expression level inhuman cancer. A, CellX tool5 was used to mine publically availablebreast, colorectal and lung cancer gene expression data from TCGA. B,RNA sequencing data analysis of TMEM219 expression in hepatocellularcarcinoma, triple negative breast cancer (TNBC), non-small cell lungcarcinoma (NSCLC), colorectal, ovarian, pancreatic cancer using theChampions TumorGraft Database provided by Champions Oncology, Inc(Hackensack, N.J.).

FIG. 2. The effect of high/low expression of TMEM219 on survival ofbreast cancer patients. Assessment of KM plot analysis shows significantsurvival difference between groups of breast cancer patients having high(n=423) and low (n=1237) TMEM219 expression (p-value=4.9E-6, HR=0.63, CI0.52-0.77).

FIG. 3A-D. Impact of IGFBP-3/IGFBP-3R expression on survival in lungcancer patients. The effect of high/low expression of TMEM219 onsurvival of lung cancer patients was assessed using the SynTarget tooland the data from the GSE30219 study. TMEM219 expression was ranked intohigh (n=162) vs. low (n=131) groups based on median expression. The logrank p-value (p-value=0.00108) was assessed. A, TMEM219: low, IGFBP3:low; B, TMEM219: high, IGFBP3: high; C, TMEM219: high, TMEM219: low; D,IGFBP3: high, IGFBP3: low.

FIG. 4. Schematic representation of the TMEM219/IGFBP-3 interaction inthe cell, a central pathway for IGFBP-3-induced inhibition of tumorgrowth, tumor angiogenesis, metastasis and chemo-resistance viainduction of apoptosis and suppression of tumor-induced NF B activity incancer cells.

FIG. 5A-F. TMEM219 (IGFBP-3R) agonist mAb Variable Region Nucleotide andAmino Acid Sequences. A, mAb #245 heavy chain (SEQ ID NO: 1); B, mAb#245 light chain (SEQ ID NO: 2); C, mAb #274 heavy chain (SEQ ID NO: 3);D, mAb #274 light chain (SEQ ID NO: 4); E, TMEM219 #274-hIgG1 chimeraheavy chain (SEQ ID NO: 5); F, TMEM219 #274-hIgG1 chimera light chain(SEQ ID NO: 6).

FIG. 6A-C. Growth Inhibitory Effects of TMEM219 Agonist mAbs in BreastCancer Cells. A, A representative western immunoblot (WIB) showingTMEM219 expression in breast cancer cells. B, Breast cells were grown upto 40% confluency and treated with 30 nM IGFBP-3R agonist mAb #2 (#274)in 1% FBS containing media for 3 days. C, Similarly breast cancer cellswere treated with different concentrations of TMEM219 agonist mAb #2(#274) for 3 days and live cells were counted using the TC20 automatedcell counter. n=3 in duplicate. MCF-10A: normal mammary epithelialcells; MCF-7, T47D: estrogen responsive breast cancer cells MDA-MB231,Hs578T: triple-negative breast cancer cells.

FIG. 7A-F. A, Anticancer effects of TMEM219 agonist mAb inbioluminescent MDA-MB-231 cells expressing TMEM219 but not inTMEM219-knockdown MDA231 cells. Treatment of TMEM219 mAb #2 (#274)results in significant activation of caspase-3, decrease of total PARPand suppression of tumor-activated NF-kB signaling (A), and cell growthinhibition (B). C, TMEM219 #274-hIgG1 chimera detects TMEM219 in MCF-7cell lysates and also D, activates caspase-3 and inhibitstumor-activated NF-kB signaling in bioluminescent MDA-MB-231 cells. E,CRISPR-CAS9-mediated knockdown of TMEM219 results in complete knockdownof TMEM219 protein expression (upper panel, sgRNA-1 & sgRNA-2) andfollowing no significant TMEM219 agonist mAb #274-induced growthinhibition (bottom panel, sgRNA-1 & sgRNA-2). F, Mechanism of Action ofTMEM219 mAbs. TMEM219 agonist mAbs specifically bind to TMEM219 andexert antitumor function in human cancers via induction ofcaspase-dependent apoptosis and suppression of tumor-activated NF-kBsignaling pathways.

FIG. 8A-C. Antitumor Effect of TMEM219 mAb in Bioluminescent OrthotopicMDA231 TNBC Mice. TMEM219 mAb #2 (#274) administration results in tumorshrinkage up to 20% (p<0.05) and 25% (p<0.01) at day 26 and day 29,respectively after tumor cell injection (A, C). No apparent body weightand damages in major organs were observed in TMEM219 mAb administratedmice (B). TMEM219 mAb #2 (#274) was intraperitoneally administrated atthe concentration of 1 mg/kg bodyweight twice per week at day 15 aftertumor cell injection.

FIG. 9A-D. TMEM219 in PDX TNBC. Expression profile of TMEM219 in PDXTNBC cells at protein (A) and mRNA levels (B) Immunohistochemicalstaining of TMEM219 expression in chemodrug-sensitive (WHIM30) andchemodrug-resistant (WHIM2) cells (C). Original magnification, ×40.Growth inhibitory effect of TMEM219 Agonist mAbs in chemodrug-sensitiveand chemodrug-resistant PDX TNBC Cells (D). Cells were treated with mAb#2 (#274) for 3 days. n=3, p/s: Radiance Photons/Second. ***, p<0.001,vs Vehicle (mouse IgG 50 nM).

FIG. 10A-D. Antitumor Effect of TMEM219 mAb in Bioluminescent OrthotopicWHIM30 PDX TNBC Mice. TMEM219 mAb #2 administration results in tumorshrinkage up to 29% at day 30 after tumor cell injection (A). Noapparent body weight and damages in major organs were observed inTMEM219 mAb administrated mice (B). Comparison of primary tumor size (C)weight (D) between mouse IgG treated control and TMEM219 mAbadministrated tumors at day 30. Mouse IgG or TMEM219 mAb #2 wasintraperitoneally administrated at the concentration of 1 mg/kgbodyweight twice per week at day 5 after tumor cell injection.

FIG. 11. IGFBP-3/TMEM219 Axis in Normal lung epithelial and NSCLC Cells.TMEM219 is expressed in normal and non-small cell lung carcinoma cellswith similar levels. However, expression of IGFBP-3 is significantlysuppressed in cancer cells compared to BEAS2B normal lung epithelialcells. More strikingly, IGFBP-3 is proteolyzed in most NSCLC cellsexcept CSCC20 cells.

FIG. 12A-E. Growth Inhibitory Effects of TMEM219 agonist mAbs in lungcancer cells (BEAS2B-NNKA) but not in normal lung epithelial cells(BEAS2B). Upper panel, NKA cells were grown up to 40% confluency; A,control; B, treated with 1 mM mAb #1 (#245) or C, 30 nM mAb # (#274) for3 days. Similarly BEAS2B-NNKA lung cancer (D) and normal lung epithelialcells (E) were treated with 1-100 nM TMEM219 agonist mAb #2 (#274) for 3days and live cells were counted using the TC20 automated cell counter.n=3 in duplicate.

FIG. 13 A-E. Therapeutic potential of AAT on CAC. AAT was treated every3 days after the last DSS cycle (day 77) for 18 days and tumors wereexamined on day 98. Macroscopic changes in colonic tissues (A) and tumorincidence (B) * p<0.05, ** p<0.01. Tissue was further processed forhematoxylin/eosin staining (C) and IHC (D). Original magnification 40×,100× respectively. (E) AAT treatment results in significant inhibitionof IGFBP-3 proteolysis in circulation.

FIG. 14A-C. Antitumor effect of TMEM219 agonist mAb #2 (#274) oncolitis-associated colon cancer in the AOM/DSS mouse model (A).Mechanism of action (B and C), TMEM219 mAb #2 (#274) inhibits coloncancer cell growth (B) and TMEM219 mAb #1 (#245) suppressesTNF-alpha-activated inflammatory NF-kappa B signaling in HT-29 coloncancer cells (C).

FIG. 15A-C. HFD fed mice with AAT administration results in increase ofinsulin sensitivity and decrease IGFBP-3 proteolysis accompanyingreduced hepatic lipogenesis and inflammation in visceral fat. A, Insulintolerance test in HFD-fed mice treated with or without AAT weekly (60mg/kg body weight) for 7 weeks. Mice were fasted for 6 h (n=3 per group)before the ITT. B, H & E staining of liver (A,C) and visceral fat (B,D)with or without AAT for 7 weeks. B, AAT treatment results in reductionin fatty liver and infiltration of monocytes in visceral fat. C, SerumIGFBP-3 proteolysis levels (ratio of IGFBP-3 fragment over totalIGFBP-3) in HFD-fed mice treated with or without AAT for 7 weeks. *P<0.05.

FIG. 16. TMEM219 mediates IGFBP-3-induced insulin sensitizing function.IGFBP-3 agonist monoclonal Abs (#245 and #274) but not non-agonist mAb(C314) inhibit TNF-a-induced suppression of glucose uptake in primaryhuman adipocytes. n=3, in duplicate; **, p<0.01

DETAILED DESCRIPTION

The present disclosure describes therapeutic agents and methods of theiruse to treat diseases involving IGFBP-3 and its receptor, IGFBP-3R, e.g.diseases and conditions caused by abnormal functioning of IGFBP-3 andIGFBP-3R. For example, in the case of diseases caused by a lack ofIGFBP-3R activation (e.g. by a lack of production or transport ofIGFBP-3) agents that bind to and activate IGFBP-3R (i.e. IGRBP-3Ragonists) are used as IGFBP-3 substitutes. As such, they are used totreat or ameliorate symptoms of cancer, metabolic syndrome, obstructiverespiratory disorders, certain inflammatory diseases, and relateddisorders. In addition, the level of expression of IGFBP-3R in tumorcells is used as an indicator of the prognosis of a cancer patient, withlow levels indicating a poor prognosis and high levels indicating arelatively good prognosis. This type of assessment allows medicalpractitioners to tailor recommended cancer treatment regimens on apatient by patient basis.

By “IGFBP-3R” or “TMEM219” refers to the Homo sapiens (human) proteinthat acts as the receptor for human “insulin-like growth factor-bindingprotein 3” or “IGFBP-3”. The receptor is also known as “transmembraneprotein 219” and is encoded by the TMEM219 gene (gene ID 124446 in theNCBI database).

By “agonist” we mean a chemical (compound, substance, etc.) that bindsto a receptor and activates the receptor to produce a biologicalresponse. In some aspects, the agonists are monoclonal antibodies (mAbs)specific for binding to the receptor “IGFBP-3R”. Upon binding, the mAbsactivate the receptor, i.e. its biological activity is elicited,increased, etc., compared to the level of activation when no agonist ornatural ligand is bound.

A “therapeutically effective amount” of a compound is an amount that issufficient to treat or prevent or ameliorate (lessen) at least onesymptom of a disease.

By “treat” or “treating” a disease, we mean that, in a treatedindividual, one or more unwanted symptoms of the disease is/areeliminated (i.e. the patient is cured), or lessened, and/or the timeinterval during which the symptoms are present is shortened, and/oronset of symptoms is delayed, compared to an untreated individual.

“Prevention” or “preventing” refers to stopping or averting (wardingoff, etc.) the occurrence of a disease or a disease aspect or symptombefore it occurs, e.g. before evidence of the disease, symptom, etc. isdetectable or measurable.

“VH CDR” refers to a heavy chain variable domain complementaritydetermining region (CDR) of an antibody. “LH CDR” refers to a lightchain variable domain CDR of an antibody. There are three CDRs (CDR1,CDR2 and CDR3), arranged non-consecutively, on the amino acid sequenceof a variable domain of an antigen receptor. Since the antigen receptorsare typically composed of two variable domains (on two differentpolypeptide chains, one heavy chain and one light chain), there are sixCDRs for each antigen receptor that can collectively come into contactwith the antigen. A single antibody molecule has two antigen receptorsand therefore contains a total of twelve CDRs, although sixty CDRs arefound on a pentameric IgM molecule.

The Agonists

The agonists described herein activate the IGFBP-3R. In some aspects,such molecules bind to the receptor, for example, at the IGFBP-3 bindingsite which is described below, specifically or selectively. The agonistsmay be of any of the many known types of molecules which bind toreceptors, for example small molecule drugs, antibodies, etc. Disclosedherein are monoclonal antibodies (mAbs) which are agonists or IGFBP-3,as well as CDRs contained within the antibodies. While the mAbs may beused to deliver the CDRs to the receptor (i.e. antibodies may be used tomediate contact between one or more CDRs and the receptor binding site)other molecules which contain one or more of the CDRs may also be usedto do so, e.g. peptides and polypeptides which comprise one or moreCDRs. Such peptides and polypeptides may be protected to decreaseproteolysis and increase bioavailability, e.g. by including“non-natural” or non-cleavable amino acids (e.g. D amino acids,norleucine norvaline, ornithine, s-benzyl cysteine, etc.) in the peptidechain, or by incorporating N-acyl groups, reduced peptide bonds, etc.,by amidating the COO⁻ terminus, by cyclization, PEGylation orglycosylation, etc. Alternatively, the agonist may be a small moleculedrug which fits the receptor binding site and binds sufficiently toactivate the receptor. By “small molecule drug” we mean an organiccompound that is of a low molecular weight (<900 daltons) and which hasa size on the order of 1 nm. Such small molecules typically bind totarget receptors via one or more of electrostatic bonding, hydrogenbonding, and/or van der Waals/London dispersion forces. In some aspects,the agonists that are not antibodies bind to or within residues 116-125of IGFBP-3R, the amino acid sequence of which is GLKGSSAGQL (SEQ ID NO:13), as described below for the monoclonal antibodies described herein.

In some aspects, the IGFBP-3R agonists that are utilized in the practiceof the present invention are monoclonal antibodies (mAbs) specific forbinding to and activating IGFBP-3R. Generally, functional studies (seeFIGS. 6, 12 and 14) indicate the ED₅₀ (effective dose, causing 50% ofmaximum effect for the measured biological effects in cells receivingthe drug) is around 20 nM. Thus, the mAbs generally exhibit an ED₅₀ inthe range of from about 1-100 nM, e.g. about 5 to 50 nM such as about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 nM or more.

The mAbs may or may not bind to the exact same residues that are boundby the natural ligand; however, they bind sufficiently to activate thereceptor. In some aspects, the antibodies bind to the same site at whichthe ligand IGFBP-3 binds. Whatever the exact position of binding, themAbs stand in for/make up for the lack of natural ligand binding andonce bound, they activate the receptor. In some aspects, the mAbs bind,for example, to portions of IGFBP-3R which are accessible and not buriedin the membrane, e.g. within residues 1-197 of IGFBP-3R. Further, insome aspects, the mAbs bind to or within residues 116-125 of IGFBP-3R,the amino acid sequence of which is GLKGSSAGQL (SEQ ID NO: 13). In otherwords, in some aspects, the mAbs bind to at least 1, 2, 3, 4, 5, 6, 7,8, or 9 consecutive amino acids within SEQ ID NO: 13, or to all 10 aminoacids of SEQ ID NO:13. In yet other aspects, the mAbs bind to from 1-9amino acids which are not consecutive in sequence, i.e. one or more(e.g. about 1-9) amino acids within this sequence do not bind directlyto the mAb. Binding to the mAbs is generally non-covalent, e.g. via oneor more of electrostatic bonding, hydrogen bonding, and/or van derWaals/London dispersion forces.

Exemplary mAbs are described herein and the sequences of exemplary mAbsare shown in FIGS. 5A-F. Those of skill in the art will recognize thatthe invention is not limited to the use of mAbs with the exact sequencesdisclosed herein. For example, conservative and/or non-conservativeamino acid substitutions may be made in the sequences as long as theresulting mAbs retain the ability to bind to and act as agonists ofIGFBP-3R. Such variants generally have at least about 50% identity tothe sequences disclosed herein, e.g. at least about 50, 55, 60, 65, 70,75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to thedisclosed sequences. In addition, antibodies may be designed andproduced which contain one or more complementarity-determining regions(CDRs) of the antibodies described herein, i.e. they contain at leastone paratope or antigen-binding region as described herein (such as atleast one CDR), but contain different non-CDR sequences. Othervariations include but are not limited to: human, humanized, or chimericantibodies, antibody fragments that bind IGFBP-3R (e.g. human IGFBP-3R),a Fab′, a F(ab′)2, a F(ab′)3, a monovalent scFv, a bivalent scFv, asingle domain antibody, etc. The antibodies may be IgG, IgM, or IgAantibodies or antigen binding fragments thereof. In addition, theantibodies may be labeled with a detectable label as described in detailbelow, or may be otherwise modified, e.g. by glycosylation. All suchvariants are encompassed by the invention, so long as the variant bindsto IGFBP-3R and acts as an agonist of IGFBP-3R.

In some aspects, the agonist is an antibody designated TMEM219 #245,TMEM219 #274 or TMEM219 #274-hIgG1 chimera. The latter is a human IgG1chimera of TMEM219 #274. The sequences of these exemplary antibodies aredepicted in FIGS. 5A-F, and Tables 1-6 show the CDR sequences of heavyand light chains of TMEM219 #245, TMEM219 #274 and TMEM219 #274-hIgG1chimera. To calculate the % identity shown in the Tables, TMEM219 #245was compared to the germline IGHV9 gene sequence for the heavy chainCDRs and to the IGKV9 gene sequence for the light chain CDRs whereasTMEM219 #274 heavy chain CDRs and light chain CDRs were compared to theIGHV9 and IGKV6 gene sequences, respectively. It is noted that the CDRsequences of TMEM219 #274-hIgG1 chimera are the same as those of TMEM219#274. It is also noted that, in an antibody, the CDRs may be denominateda first, second, third, etc. antibody as required for clarity.

TABLE 1 CDR sequences of TMEM219#245, VHDJH heavy chain (IgG2a)Identity(%) compared  Mis- to IGHV9 From To Length Matches matches geneSequences CDR1- 70 93 24 22 2 91.7 DNA: GGG TTT ACC TTC IMGTACA TAC TAT GGA  (SEQ ID NO: 14) Protein:  G F T F T Y Y G(SEQ ID NO: 15) CDR2- 145 168 24 24 0 100 DNA: ATA AAC ACC TAC IMGTACT GGA GAG CCA  (SEQ ID NO: 16) Protein:  I N T Y T G E P(SEQ ID NO: 17) CDR3- 283 288 6 6 0 100 DNA: GCA AGA GGG CGT MGTACG GTA GTG GGC TTT  germ- GAC TCT line (SEQ ID NO: 18) Protein: A R G R T V V G F D S (SEQ ID NO: 19)

TABLE 2 CDR sequences of TMEM219#245, VKJK light chain Identity(%)Compared Mis- to IGKV9 From To Length Matches matches gene SequencesCDR1-  79 96 18 18 0 100 DNA: CAG GAC ATT  IMGT GGT AGT AGC (SEQ ID NO: 20) Protein:  Q D I G S S (SEQ ID NO: 21) CDR2-  148 156 9 90 100 DNA: GCC ACA TCC IMGT Protein: A T S CDR3- 265 284 20 20 0 100DNA: CTA CAA TAT  MGT GCT AGT TCT CCG  germ- TAC ACG line(SEQ ID NO: 22) Protein:  L Q Y A S S P (SEQ ID NO: 23)

TABLE 3 CDR sequences of TMEM219#274, VHDJH heavy chain (IgG3)Identity(%) Compared Mis- to IGHV9 From To Length Matches matches geneSequences CDR1- 76 99 24 23 1 95.8 DNA: GGG TAT ACT  IMGTTTC ACA AAC TAT GGA  (SEQ ID NO: 24) Protein:  G Y T F T N Y G(SEQ ID NO: 25) CDR2-  151 174 24 21 3 87.5 DNA: ATA AAC ACC  IMGTTAC ACC AGA GAG ACA  (SEQ ID NO: 26) Protein:  I N T Y T R E T(SEQ ID NO: 27) CDR3- 289 294 6 6 0 100 DNA: GCA AGA GGG  MGTTCT ACG ATG TAT  germ- GGT CTG GAC AAG line (SEQ ID NO: 28) Protein: A R G S T M Y G L D K (SEQ ID NO: 29)

TABLE 4 CDR sequences of TMEM219#274, VKJK light chain Identity(%)Compared Mis- to IgKV6 From To Length Matches matches gene SequencesCDR1- 79 96 18 18 0 100 DNA: CAG AAT  IMGT GTG GGT ACT AAT (SEQ ID NO: 30) Protein:  Q N V G T N (SEQ ID NO: 31) CDR2- 148 156 9 90 100 DNA: TCG GCA TCC IMGT Protein: S A S CDR3- 265 287 23 22 1 95.7DNA: CAC CAA  MGT TAT AAC AGC TAT germ- CCT CTC ACG line (SEQ ID NO: 32)Protein: H Q Y N S Y P L T (SEQ ID NO: 33)

TABLE 5 TMEM219 #274-hIgG1 chimera CDR sequences(same as those of TMEM219@274 Heavy chain κ Light chain CDR1-  Protein: Protein: Q N V G T N IMGT G Y T F T N Y G (SEQ ID NO: 25)(SEQ ID NO: 31) CDR2-  Protein:  Protein: S A S IMGT I N T Y T R E T(SEQ ID NO: 27) CDR3-  Protein:  Protein:  MGT A R G S T M Y H Q Y N S Y P L T germ- G L D K (SEQ ID NO: 33) line (SEQ ID NO: 29)

In some aspects, the antibody comprises one or more of the CDRs ofantibody TMEM219 mAb #1 (#245) as follows: (a) a VH CDR at least 90%identical to SEQ ID NO: 15; (b) a VH CDR at least 90% identical to SEQID NO: 17; (c) a VH CDR at least 90% identical to SEQ ID NO: 19; (d) aVL CDR at least 90% identical to VL CDR1 of SEQ ID NO: 21; (e) a VL CDRat least 90% identical to SEQ ID NO: 23; and (f) a VL CDR at least 90%identical to the sequence ATS. That is, the sequence is about 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or even 100% identical to the indicatedsequence.

In other aspects, the antibody comprises one or more of the CDRs ofantibody TMEM219 mAb #2 (#274) as follows: (a) a VH CDR at least 90%identical to SEQ ID NO: 25; (b) a VH CDR at least 90% identical to SEQID NO: 27; (c) a VH CDR at least 90% identical to SEQ ID NO: 29; (d) aVL CDR at least 90% identical to VL CDR1 of SEQ ID NO: 31; (e) a VL CDRat least 90% identical to the sequence SAS; and (f) a VL CDR at least90% identical to SEQ ID NO: 33. That is, the sequence is about 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or even 100% identical to the indicatedsequence.

In some aspects, the antibody comprises one or more of the CDRs ofantibody TMEM219 #274-human IgG1 (#274-hIgG1) chimera as follows: (a) aVH CDR at least 90% identical to SEQ ID NO: 25; (b) a VH CDR at least90% identical to SEQ ID NO: 27; (c) a VH CDR at least 90% identical toSEQ ID NO: 29; (d) a VL CDR at least 90% identical to VL CDR1 of SEQ IDNO: 31; (e) a VL CDR at least 90% identical to the sequence SAS; and (f)a VL CDR at least 90% identical to SEQ ID NO: 33. That is, the sequenceis about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or even 100% identicalto the indicated sequence.

Those of skill in the art will recognize that suitable antibodies foruse in the methods described herein are obtainable from hybridomas usingtechnology that is known in the art. Alternatively, the antibodies maybe made by recombinant technology, e.g. by cell culture or bacterialculture, as is well-known in the art, or even synthetically via chemicalpeptide synthesis.

In some aspects, the antibodies are used to treat diseases such ascancer. In these aspects, the antibodies may be modified to includeother effector molecules. Non-limiting examples of effector moleculesthat can be attached to antibodies include toxins, therapeutic enzymes,antibiotics, radio-labeled nucleotides and the like. As is known in theart, linking molecules may be used to join the antibody to the effectormolecule. Such effectors may be especially useful when the disease thatis treated is cancer.

In addition, the encoding nucleic acids need not be identical to thosedepicted in FIG. 5A-F, e.g. due to the redundancy of the genetic code.In general, encoding sequences will generate the antibodies describedherein, and may or may not be codon optimized for production in aparticular way, e.g. in plant, mammalian or bacterial host cells.Nucleic acids encompassed by the invention include but are not limitedto DNA and RNA, and sequences that are at least about 90% homologous tothe sequences disclosed herein (e.g. about 90, 91, 92, 93, 94, 95, 96,97, 98, 99 or even 100% homologous). Further, the invention encompassesvectors (plasmids, cosmids, viral vectors, etc.) which include theencoding nucleic acids sequences, and cells which contain the nucleicacid sequences and/or the vectors.

Compositions

The invention also comprises compositions comprising one or more of theagonists (e.g. antibodies) described herein. Those of skill in the artwill recognize that the components in the compositions will varydepending on whether the antibodies are used in diagnostic methods or intreatment methods, whether or not they are labeled, etc.

When used for treatment methods, the compounds described herein aregenerally delivered (administered) as a pharmaceuticalcomposition/formulation. The compositions generally include one or moresubstantially purified antibodies as described herein, and apharmacologically suitable (physiologically compatible) carrier, whichmay be aqueous or oil-based. In some aspects, such compositions areprepared as liquid solutions or suspensions, or as solid forms such astablets, pills, powders and the like. Solid forms suitable for solutionin, or suspension in, liquids prior to administration are alsocontemplated (e.g. lyophilized forms of the compounds), as areemulsified preparations. In some aspects, the liquid formulations areaqueous or oil-based suspensions or solutions. In some aspects, theactive ingredients are mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredients, e.g.pharmaceutically acceptable salts. Suitable excipients include, forexample, water, saline, dextrose, glycerol, ethanol and the like, orcombinations thereof. In addition, the composition may contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, preservatives, and the like. If it is desired toadminister an oral form of the composition, various thickeners,flavorings, diluents, emulsifiers, dispersing aids or binders and thelike are added. The composition of the present invention may contain anysuch additional ingredients so as to provide the composition in a formsuitable for administration. The final amount of antibody in theformulations varies, but is generally from about 1-99%. Still othersuitable formulations for use in the present invention are found, forexample in Remington's Pharmaceutical Sciences, 22nd ed. (2012; eds.Allen, Adejarem Desselle and Felton).

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins (such as humanserum albumin), buffer substances (such as twin 80, phosphates, glycine,sorbic acid, or potassium sorbate), partial glyceride mixtures ofsaturated vegetable fatty acids, water, salts or electrolytes (such asprotamine sulfate, disodium hydrogen phosphate, potassium hydrogenphosphate, sodium chloride, or zinc salts), colloidal silica, magnesiumtrisilicate, polyvinyl pyrrolidone, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, methylcellulose,hydroxypropyl methylcellulose, wool fat, sugars such as lactose, glucoseand sucrose; starches such as corn starch and potato starch; celluloseand its derivatives such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients such as cocoa butter and suppository waxes; oils suchas peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil;corn oil and soybean oil; glycols; such a propylene glycol orpolyethylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

“Pharmaceutically acceptable salts” refers to the relatively non-toxic,inorganic and organic acid addition salts, and base addition salts, ofcompounds of the present invention. These: salts can be prepared in situduring the final isolation and purification of the compounds. Inparticular, acid addition salts can be prepared by separately reactingthe purified compound in its free base form with a suitable organic orinorganic acid and isolating the salt thus formed. Exemplary acidaddition salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate,palmitate, stearate, laurate, borate, benzoate, lactate, phosphate,tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate,mesylate, glucoheptonate, lactiobionate, sulfamates, malonates,salicylates, propionates, methylene-bis-.beta.-hydroxynaphthoates,gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates,ethanesulfonates, benzenesulfonates, p-toluenesulfonates,cyclohexylsulfamates and laurylsulfonate salts, and the like. See, forexample S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 66,1-19 (1977) which is incorporated herein by reference. Base additionsalts can also be prepared by separately reacting the purified compoundin its acid form with a suitable organic or inorganic base and isolatingthe salt thus formed. Base addition salts include pharmaceuticallyacceptable metal and amine salts. Suitable metal salts include thesodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts.The sodium and potassium salts are preferred. Suitable inorganic baseaddition salts are prepared from metal bases which include sodiumhydride, sodium hydroxide, potassium hydroxide, calcium hydroxide,aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinchydroxide and the like. Suitable amine base addition salts are preparedfrom amines which have sufficient basicity to form a stable salt, andpreferably include those amines which are frequently used in medicinalchemistry because of their low toxicity and acceptability for medicaluse ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine,ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine,diethanolamine, procaine, N-benzylphenethylamine, diethylamine,piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammoniumhydroxide, triethylamine, dibenzylamine, ephenamine,dehydroabietylamine, N-ethylpiperidine, benzylamine,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, ethylamine, basic amino acids, e.g., lysine andarginine, and dicyclohexylamine, and the like.

The compositions may be administered in vivo by any suitable routeincluding but not limited to: inoculation or injection (e.g.intravenous, intraperitoneal, intramuscular, subcutaneous, intra-aural,intraarticular, intramammary, intratumoral, and the like), by topicalapplication and by absorption through epithelial or mucocutaneouslinings (e.g., nasal, oral, vaginal, rectal, gastrointestinal mucosa,and the like). Other suitable means include but are not limited to:inhalation (e.g. as a mist or spray), orally (e.g. as a pill, capsule,liquid, etc.), intravaginally, intranasally, rectally, etc. In preferredembodiments, the mode of administration is oral or by injection. Inaddition, the compositions may be administered in conjunction with othertreatment modalities such as substances that boost the immune system,various chemotherapeutic agents, antibiotic agents, and the like.

The dose of antibody that is administered varies according to factorssuch as the exact type of disease, the method of administration, overallhealth of the patient, etc., but is generally in the range of from about1-100 mg/kg, or from about 2.5-75 mg/kg or 5-50 mg/kg of body weight,including all whole number and decimal fractions thereof lying withinthe ranges.

Diagnostics

In some aspects, the antibodies described herein are used to detect theexpression level of IGFBP-3R (TMEM219) to diagnose and prognose cancer.In such cases, the antibodies may be labeled with a detectable reportermolecule. A reporter molecule is defined herein as any moiety that maybe detected using an assay. Non-limiting examples of reporter moleculesthat may be conjugated to antibodies include but are not limited toenzymes, radiolabels, haptens, fluorescent labels, phosphorescentmolecules, chemiluminescent molecules, chromophores, luminescentmolecules, photoaffinity molecules, secondary or tertiary antibodies,and colored particles or ligands, such as biotin. As is known in theart, linking molecules may be used to join the antibody to the reportermolecule. In further aspects, the antibodies may be immobilized on asolid support for use in an assay, e.g. on beads, in the wells of anassay plate, etc.

Compositions for diagnostic agents can include any of the componentslisted above for compositions, but less care needs to be taken topromote physiological compatibility. Generally the assay solution isaqueous based and is buffered, and may contain preservatives, varioussalts, etc. Kits comprising a container comprising the antibodies arealso provided. The kits may contain other reagents (e.g. reagents todetect a detectable label), directions for use, positive and/or negativereference standards, etc.

Diagnostic assays generally involve obtaining a biological sample ofinterest from a subject (e.g. a blood or plasma sample, a tissue sample,a biopsy sample, etc.) and exposing the sample to one or more antibodiesas disclosed herein under conditions which allow binding to the antibodyto a target molecule of interest, e.g. the TMEM219 molecule. Theantibodies generally comprise a detectable label, which after binding tothe molecule of interest, is detected by methods known in the art andspecific for each different label, and the amount of labeled antibody iscorrelated to the amount of the molecule of interest that is present inthe sample, e.g. by the use of one or more reference values, asdescribed below.

Prognosing and Treating Cancer

The methods described herein are used to diagnose cancer and/or toconfirm a cancer diagnosis and/or to determine the prognosis of apatient with cancer, e.g. to predict one or more of metastaticpotential, chances of recurrence and prospects for survival. In someaspects, the patients treatment is tailored (modified, selected, etc.)according to the results of the method. Cancers which may be assessed inthis manner include but are not limited to breast cancer, colon cancer,lung cancer, ovarian cancer, pancreatic cancer, liver cancer andleukemia.

The invention thus provides methods of determining the prognosis of asubject with cancer and treating the subject accordingly, comprising: i)measuring a level of TMEM219 expression in a tumor sample (e.g. a biopsysample) from the subject; ii) comparing the level of TMEM219 expressionobtained in step i) with a reference level of TMEM219 expression; andiii) if the level of TMEM219 expression is the same or lower than thereference level of TMEM219 expression, then, iv) concluding that thepatient has a poor prognosis and providing aggressive anti-cancertreatment to the patient, examples of which include combination therapywith TMEM219 agonist antibody and chemotherapy, radiotherapy, adjuvanttherapy or hormone therapy or v) if the level of TMEM219 expression ishigher than the reference level of TMEM219 expression, then iv)concluding that the patient has a good prognosis and providing lessaggressive anti-cancer treatment to the patient, examples of whichinclude combination therapy with TMEM219 agonist antibody andchemotherapy or radiotherapy. The level of TMEM219 expression in thetumor sample is determined by any of many known methods for determiningprotein expression, including but not limited to: measuring the proteinper se e.g. using antibodies (which are generally labelled with adetectable label), etc.; measuring mRNA encoding the protein, e.g. viaPCR using primers (which are generally labelled with a detectablelabel), etc.

Once the level of TMEM219 expression has been measured, the level iscompared to at least one reference value. Establishing suitablereference values is known in the art. Generally, such a value isestablished by measuring a substance of interest (protein, mRNA, etc.)that is indicative of the amount of TMEM219 expression in one or moreappropriate control groups of comparable individuals who are healthy,i.e. in the present case, individuals who do not have cancer. However,additional reference values may also be used, e.g. reference valuesestablished using tissue from other cancer patients with high and/or lowlevels of TMEM219 expression, and reference values based on cancerousand/or non-cancerous tissue from the patient him/herself. Referencevalues may be obtained from patients with cancer or who have had cancerand who have or have not been treated for cancer, etc. e.g. patients inremission, those being actively treated for cancer, and the like.Controls may or may not be matched with respect to e.g. age, gender,ethnicity, overall health, life style, etc., as appropriate.

As used herein, a level of TMEM219 expression that is “equal to”,“higher than” or “lower than” a reference value is: the same as thereference value (e.g. +/− about 5-10% of the reference value), or higherthan the reference value e.g. at least about 10% or more higher than thereference value, or lower than the reference value e.g. at least about10% or more lower than the reference value, respectively. In someaspects, an amount of mRNA that is “equal to” is within +/− about 1-5%of Log [Fragments Per Kilobase of transcript per Million (RPKM)+1] 6.06for colorectal cancer, 5.80 for ovarian cancer, 5.93 for pancreaticcancer, 5.43 for hepatocellular carcinoma, 5.77 for NSCLC and 5.91 forTNBC. A “higher” level is a value that is at least about 5% greater thatthe above mentioned Log(RPKM+1) values. A “lower” level is a value thatis at least about 5% less than the above mentioned Log(RPKM+1) values.For example, for colorectal cancer, an “equal” value falls within arange of from about 6.363 to about 5.757, a high value exceeds 6.363 anda low value is below 5.757. Alternatively, the “equal” range may bewithin 1-10% of the indicated values, low values are at least 1-10%below and high values are at least 1-10% above the indicated referencevalues. Patients that are found to have TMEM219 expression levels equalto or lower than a suitable corresponding reference value are consideredto have a poor prognosis, e.g. a high likelihood of one or more ofmetastasis, recurrence, and low overall survival. Such patients aretreated with an aggressive treatment regimen, as described below.Patients that are found to have TMEM219 expression levels higher thanthe reference value are considered to have a good prognosis, e.g. a lowlikelihood of one or more of metastasis, recurrence, and overall highexpectation of survival. Such patients are treated with a lessaggressive treatment regimen, as described below, and can avoidsuffering the unwanted, detrimental side effects of aggressivetreatment. Such patients may in fact need no therapy (or no furthertherapy, if they have already been treated) but may benefit frommonitoring the level of TMEM219 expression on an ongoing basis.

It is within the purview of the skilled medical practitioner to selectan appropriate therapeutic regimen. Therapeutic regimens may becomprised of the use of cancer chemotherapeutic agents and/or radiationand/or surgery. A cancer chemotherapeutic agent is a chemical compoundor biological agent that retards, slows, or stops the growth of canceror is approved to treat cancer by the U.S. Food and Drug Administration.Examples of cancer chemotherapeutic agents include, but are not limitedto: paclitaxel, docetaxel, imatinib mesylate, sunitinib malate,cisplatin, etoposide, vinblastine, methotrexate, adriamycin,cyclophosphamide, doxorubicin, daunomycin, 5-fluoruracil, vincristine,endostatin, angiostatin, bevacizumab, and rituximab. Another example ofa cancer treatment agent is radiation. Thus, the cancer treatment maycomprise radiotherapy, fractionated radiotherapy, chemotherapy, orchemo-radiotherapy (a combination of one or more chemotherapeutic agentsand radiation). “Biological” anti-cancer agents include e.g. antibodies,proteins, RNA, siRNA, single guide RNA (sgRNA), DNA, etc.

As defined herein, an “aggressive cancer treatment” or “aggressivecancer treatment regimen” is generally determined by a medicalprofessional such as a physician and/or radiologist and can be specificfor each patient. In some aspects, an aggressive cancer treatmentregimen is as defined by the National Comprehensive Cancer Network(NCCN), and has been defined in the NCCN Guidelines™ as including one ormore of 1) intensified imaging (CT scan, PET/CT, MRI, chest X-ray), 2)discussion and/or offering of sentinel lymph node biopsy with subsequentpartial or complete lymphadenectomy, 3) inclusion in ongoing clinicaltrials, and 4) therapeutic intervention with interferon alpha treatmentand radiation to nodal basin. In general, the phrase “aggressive cancertreatment” refers to a cancer treatment, or combination of treatments,and/or a chemotherapy regimen that is effective for treating the targetcancer tumor or cell, but is associated with or known to cause highertoxicity and more side effects than another type of treatment for thespecified cancer type. Aggressive treatment may include one or more ofsurgical intervention, chemotherapy, radiation therapy, adjuvanttherapy, hormone therapy, close clinical surveillance, etc. Aggressivetreatment may comprise proactive treatment to reduce or preventmetastasis, including distant or multiple metastases, e.g. usingsystemic chemotherapy. For aggressive treatment, exceptionally toxicchemotherapeutic agents may be preferred, as may higher and/or morefrequent doses of one or more anti-cancer agents, and/or a longer courseduration of therapy (chemotherapy, radiation, etc.), and/or a repetitionof therapy. For example, a radical mastectomy may be recommended,together with lymph node removal, chemotherapy and radiation for abreast cancer patient.

Less aggressive treatment may also comprise surgical intervention,chemotherapy, radiation therapy, adjuvant therapy, hormone therapy, orclose clinical surveillance, etc. It may also comprise proactivetreatment to reduce or prevent local, organ-specific, tissue specific,or site-specific metastasis. However, in general, the treatment may bemore localized and focus on the primary tumor, using e.g. resectionfollowed by targeted drug therapy, such as treatment using antibodieswhich target the particular tumor type, or an implanted radiationsource, etc. For breast cancer, a lumpectomy may be recommended toremove a cancerous breast tumor, preceded by neo-adjuvant treatment toshrink the tumor prior to surgery, rather than a radical mastectomy. Ifa course of radiation is prescribed, it may be a shorter and/or lessintense course than that which is recommended for aggressive treatment.

In general, one of skill in the art will be able to determine if acancer treatment, combination of treatments, or chemotherapy regimen isless or more, and this may vary by cancer type, the age and generalphysical health of the patient, etc. For example, a less aggressivetreatment may include adjuvant chemotherapy comprising surgicalresection of the primary tumor and a chemotherapy regimen comprising5-FU, leucovorin and bevacizumab, while a more aggressive cancertreatment may include adjuvant chemotherapy comprising surgicalresection and a chemotherapy regimen comprising FOLFOX and BV, and themost aggressive cancer treatment may include surgical resection and achemotherapy regime comprising Irinotecan and Cetuximab.

In addition, some subjects who have not been diagnosed with cancer butwho are at high risk of developing cancer may benefit from monitoringTMEM219 expression on an ongoing basis. Examples of such subjectsinclude but are not limited to subject with a genetic predisposition todevelop cancer (e.g. women with mutations in one or both of the BRCA1and BRCA2 genes), or who have experienced an environmental insult thatmay result in cancer (e.g. exposure to radiation, inhalation of toxicparticles, contact with carcinogenic chemicals, etc.) or who have or areengaged in high risk activities with respect to cancer such as smoking.Such subject may be monitored on an ongoing basis by determining TMEM219expression levels over an extended period of time (months or years) andby a comparison of early, non-symptomatic levels in a tissue of interest(e.g. breast or lung tissue) to levels measured over time, or to arelevant reference value. In this manner, the development of canceroustissues may be detected and early treatment can begin.

Cancer Treatment

In other aspects, the present disclosure also provides IGFBP-3R agonistsfor use in the treatment of cancer. The patient may or may not have beendiagnosed using the methods described in the preceding section. Theagonists advantageously cause cancer cell death without killing normal,non-tumor cells. Generally, the IGFBP-3R agonists are mAbs as disclosedherein, and the methods involve preventing or treating cancer byadministering a therapeutically effective amount of at least one ofagonist of the IGFBP-3R, such as a mAb disclosed herein.

In some aspects, the antibodies are used in single-agent therapy. Inother aspects, the antibodies are used in combinatorial antitumoractivity (which may give additive or synergistic results) with otherchemotherapeutic agents including but are not limited to iniparib,gemcitabine, onartuzumab carboplatin, cisplatin, paclitaxel, bortezomib,erlotinib, everolimus, synribo, etoposide, doxorubicin, venetoclax,navitoclax, nivolumab and pembrolizumab. In addition, the agonists maybe used in combination with other cancer therapies such as radiation,surgery/resection, In some aspects, the cancer that is treated is breastcancer (e.g. TNBC), colon cancer, lung cancer, ovarian cancer,pancreatic cancer, head and neck cancer, prostate cancer, liver canceror a liquid tumor (e.g. a leukemia), etc.

For the treatment of cancer, the amount of antibody that is administeredis generally in the range of from about 1-100 mg/kg, and is preferablyfrom about 5 to 50 mg/kg, e.g. about 5, 10, 15, 20, 25, 30, 35, 40, 45or 50 mg/kg.

Metabolic Syndrome

“Metabolic syndrome”, a cluster of conditions which occur together, isknown to increase the risk of heart disease, stroke and type 2 diabetes.In fact, Type 2 diabetes is sometimes considered a “complication” ofmetabolic syndrome, and “pre-diabetes” and metabolic syndrome areconsidered by some to be the same disease. Although diagnosticguidelines differ among e.g. the American Heart Association, the WorldHealth Organization, and other institutions, metabolic syndrome isgenerally diagnosed in a subject when e.g. at least 3 of the followingconditions are present: increased or high blood pressure, high bloodsugar, excess body fat around the waist, abnormally low HDL cholesteroland high triglyceride levels.

“High blood pressure” generally refers to blood pressure of 140 systolicor higher and/or 90 diastolic or higher that stays high over time. Insome aspects, blood pressure of 130/85 or more is taken as diagnosticfor metabolic syndrome, when taken together with e.g. at least 2 otherrelevant symptoms.

“High blood sugar” generally refers to a blood sugar that is higher than130 mg/dL (milligrams per deciliter) after not eating or drinking for atleast 8 hours, although in some aspects a fasting blood glucose level of100 mg/dl or above is taken as diagnostic for metabolic syndrome, whentaken together with other relevant symptoms. High blood sugar isgenerally caused by insulin resistance, and insulin resistance may beincluded as a hallmark of metabolic disease.

Central obesity (also known as abdominal, visceral, male-pattern orapple-shaped adiposity) and/or the presence of ectopic fat arediagnostic criteria for metabolic syndrome. Central obesity generallyrefers to a large waist circumference e.g. for a man, a waistcircumference of at least 40 inches (102 centimeters) and for a woman atleast 35 inches (89 centimeters). For ethnic Asian Americans, the cutoffvalues are >90 cm (35 in) in men or >80 cm (32 in) in women. Ectopic fatrefers to fat deposition in organs/tissues that do not normally storefat.

A fasting HDL cholesterol level of 40 mg/dl or lower in men and 50 mg/dlor lower in women (dyslipidemia) is a hallmark of metabolic disorder.This may also be referred to as abnormal (high) cholesterol, lipiddisorder, dyslipidemia, hyperlipidemia, or hypercholesterolemia, etc.Some practitioners use total (HDL and LDL) cholesterol levels, which areconsidered high at levels of more than 200 mg/dL, e.g. 200 to 239 mg/dLmay be referred to as “borderline high,” and 240 mg/dL or more may beconsidered “high”.

“High triglyceride levels” (dyslipidemia) generally refers totriglyceride levels that are at least higher than 100 milligrams perdeciliter of blood (mg/dL), with borderline high levels being 150 to 199mg/dL, high levels being 200 to 499 mg/dL and very high levels being 500mg/dL and above. According to some guidelines, serum triglycerides of150 mg/dl or above are considered diagnostic, when taken together withat least 2 other relevant symptoms.

Based on the guidelines from the National Heart, Lung, and BloodInstitute (NHLBI) and the American Heart Association (AHA), any three ofthe preceding traits in the same individual meet the criteria for adiagnosis of metabolic syndrome. In addition, it is noted that insulinresistance, metabolic syndrome, and prediabetes are closely related toone another and have overlapping aspects, and each of these may betreated or prevented by the practice of the methods described herein.

In particular, insulin resistance is a syndrome (a set of signs andsymptoms) that is also part of the larger constellation of symptomscalled the metabolic syndrome. Insulin resistance (IR) is a pathologicalcondition in which cells fail to respond normally to the hormoneinsulin. Normally, the body produces insulin when glucose starts to bereleased into the bloodstream from the digestion of carbohydrates in thediet, and this insulin response triggers glucose being taken into bodycells, to be used for energy, and inhibits the body from using fat forenergy. The concentration of glucose in the blood decreases as a result,staying within the normal range even when a large amount ofcarbohydrates is consumed. When the body produces insulin underconditions of insulin resistance, the cells are resistant to the insulinand are unable to use it as effectively, leading to high blood sugar.Beta cells in the pancreas subsequently increase their production ofinsulin, further contributing to a high blood insulin level. People whodevelop type 2 diabetes usually pass through earlier stages of insulinresistance and prediabetes. Insulin resistance may also develop inpatients who have recently experienced abdominal or bariatricprocedures, although this acute form of post-operative insulinresistance tends to be short term.

The present disclosure provides IGFBP-3R agonists for use in theprevention and treatment of metabolic syndrome, i.e. one or more of thesymptoms, signs or criteria used for diagnosing metabolic syndrome isprevented or treated by administering one or more of the agonistsdescribed herein, including insulin resistance. In some aspects, theagonists are monoclonal antibodies (mAbs) specific for binding to thereceptor “IGFBP-3R” as disclosed herein. Thus, the agonists are used toprevent or treat metabolic syndrome and/or complications thereof, and/ordiseases caused by metabolic syndrome, e.g. to prevent symptoms fromworsening and/or causing even more serious conditions such as heartdisease, stroke and type 2 diabetes.

For the treatment of metabolic syndrome, the amount of antibody that isadministered is generally in the range of from about 1-100 mg/kg, and ispreferably from about 5 to 50 mg/kg, e.g. about 5, 10, 15, 20, 25, 30,35, 40, 45 or 50 mg/kg.

Obstructive Respiratory Diseases

Obstructive lung disease is a category of respiratory diseasecharacterized by airway obstruction. Many obstructive diseases of thelung result from narrowing of the smaller bronchi and largerbronchioles, often because of excessive contraction of the smooth muscleitself. It is generally characterized by inflamed and easily collapsibleairways, obstruction to airflow, problems exhaling and frequent medicalclinic visits and hospitalizations. Types of obstructive lung diseaseinclude; asthma, bronchiectasis, bronchitis and chronic obstructivepulmonary disease (COPD). Although COPD shares similar characteristicswith all other obstructive lung diseases, such as the signs of coughingand wheezing, they are distinct conditions in terms of disease onset,frequency of symptoms and reversibility of airway obstruction. Cysticfibrosis is also sometimes included in obstructive pulmonary disease.

Obstructive respiratory disorders include but are not limited to: Asthma(including exercise-induced asthma, occupational asthma and nocturnalasthma. Asthma is characterized by hyperresponsive bronchial tubes(airways) that become inflamed and produce excess mucus. The musclesaround the airways tighten making the airways narrower. Asthma isusually triggered by e.g. dust or pollen that produces an allergicreaction, but may also be triggered by an upper respiratory tractinfection, cold air, exercise or smoke. Asthma causes recurring episodesof wheezing, breathlessness, chest tightness, and coughing.Bronchiectasis, which refers to the abnormal, irreversible dilatation ofthe bronchi caused by destructive and inflammatory changes in the airwaywalls. Bronchiectasis has three major anatomical patterns: cylindricalbronchiectasis, varicose bronchiectasis and cystic bronchiectasis.

Chronic obstructive pulmonary disease, a term that includes theconditions emphysema and chronic bronchitis. Most patients with COPDhave characteristics of both conditions to varying degrees and typicallythe symptoms are irreversible. However, many COPD patients have somedegree of reversibility in their airways.

The subjects who are treated using the methods described herein havegenerally been diagnosed with one or more obstructive respiratorydiseases. Identifying such subjects depends on several factors and onthe exact disease being diagnosed. However, one commonality amongpatients is an FEV1/FVC ratio of less than 0.7, i.e. the inability toexhale 70% of their breath within one second.

For the treatment of an obstructive respiratory disease, the amount ofantibody that is administered is generally in the range of from about1-100 mg/kg, and is preferably from about 5 to 50 mg/kg, e.g. about 5,10, 15, 20, 25, 30, 35, 40, 45 or 50 mg/kg.

Inflammatory Disorders

In other aspects, the compositions and method described herein are usedto prevent or treat inflammatory disorders. Inflammatory disordersgenerally result when the immune system attacks the body's own cells ortissues (auto-inflammatory diseases), causing chronic abnormalinflammation and resulting in chronic pain, redness, swelling,stiffness, and damage to normal tissues.

Exemplary inflammatory disorders that may be prevented/treated includebut are not limited to: ulcerative colitis, colitis, Crohn's disease,atherosclerosis, chronic peptic ulcer, chronic obstructive lung disease,idiopathic pulmonary fibrosis, tuberculosis, arthritis (osteoarthritis,rheumatoid arthritis (RA), psoriatic arthritis), chronic sinusitis,asthma, hepatitis, ankylosing spondylitis, liver fibrosis, non-alcoholicsteatohepatisis and chronic periodontitis.

For the treatment of inflammation and/or an inflammatory disorder, theamount of antibody that is administered is generally in the range offrom about 1-100 mg/kg, and is preferably from about 5 to 50 mg/kg, e.g.about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mg/kg.

EXAMPLES Example 1. Diagnostics: TMEM219 as a Molecular Marker forPredicting Recurrence and Survival in Cancer

Bioinformatics analysis of The Cancer Genome Atlas (TCGA) data showedthat IGFBP-3 is significantly lower in breast cancer, whereas expressionof TMEM219 is similar or higher in breast, colon and lung cancerscompared to normal tissues. This finding shows that the antitumorIGFBP-3/TMEM219 axis is impaired during tumorigensis/progression ofcancer due to suppressed expression of IGFBP-3 but not its receptorTMEM219 (FIG. 1). Further assessment using data from the METABRIC study(Curtis et al., Nature. 2012:486(7403):346-52) and the SynTarget tool[Pub Med PMID: 26915292] showed that TMEM219 expression is stronglycorrelated with survival of breast cancer (Table 1).

TABLE 1 Assessment of Correlation of TMEM219 with Survival of BreastCancer Patients using the SynTarget tool Cancer Subtype Survival(Clinical Variable) Gene ID (Probe ID) Effect p-value All samplesTMEM219 Pos 5.32e−07 (ILMN_1737644) Histological_type = “IDC” TMEM219Pos 1.58e−06 (ILMN_1737644) HER2_SNP6_state = TMEM219 Pos 3.95e−06“NEUT” (ILMN_1737644) Menopausal_status_ TMEM219 Pos  4.38−05 inferred =“post” (ILMN_1737644) Her2.Expr = “-“ TMEM219 Pos 7.31e−05(ILMN_1737644) HER_IHC_status = “null” TMEM219 Pos 0.000146(ILMN_1737644) ER.Expr = “+” TMEM219 Pos 0.000569 (ILMN_1737644) PR.Expr= “+” TMEM219 Pos 0.000652 (ILMN_1737644) HER2_IHC_status = “1” TMEM219Pos 0.000655 (ILMN_1737644) ER_IHC_status = “pos” TMEM219 Pos 0.000817(ILMN_1737644) Site “2” TMEM219 Pos 0.00102 (ILMN_1737644) Celluarlity =“high” TMEM219 Pos 0.00107 (ILMN_1737644) Menopausal_status_ TMEM219 Pos0.00121 inferred = “pre” (ILMN_1737644) Stage = “null” TMEM219 Pos0.00123 (ILMN_1737644) Grade = “3” TMEM219 Pos 0.00144 (ILMN_1737644)Stage = “2” TMEM219 Pos 0.00174 (ILMN_1737644) Cellularity = “moderate”TMEM219 Pos 0.00257 (ILMN_1737644) Lymph_nodes_positive = TMEM219 Pos0.00559 “2” (ILMN_1737644) Grade = “2” TMEM219 Pos 0.00608(ILMN_1737644) P53_mutation_status = TMEM219 Pos 0.00809 “WT”(ILMN_1737644)These results were confirmed using the Kaplan-Meyer survival analysis,which detected a significant survival difference between groups ofpatients having high (n=423) and low (n=1237) TMEM219 expression[p-value=4.9E-6, hazard ratio (HR)=0.63, confidence interval (CI)0.52-0.77)] (FIG. 2). This strong correlation is also observed in colonand lung cancer (Table 2, FIG. 3).

TABLE 2 Assessment for Correlation of TMEM219 with Survival of Colon andLung Cancer Patient using SynTarget Tool Cancer Subtype GEO Cancer(Clinical Survival Dataset ID Type Variable) Gene IS (Probe ID) Effectp-value GSE30219 Lung all samples TMEM219(224981_AT) positive 0.0010GSE30219 Lung gender = F TMEM219(224981_AT) positive 0.0074 GSE39582Colon tnm.stage = 1 TMEM219(228513_AT) positive 0.0094 GSE39582 Coloncit.molecular TMEM219(228513_AT) positive 0.018 subtype = C5 GSE39582Colon tumor.location = TMEM219(228513_AT) positive 0.021 distal GSE39582Colon mmr.status = TMEM219(228513_AT) positive 0.025 pMMR

These findings provide solid evidence for TMEM219 as a molecular markerfor predicting recurrence and survival in breast, colon and lung cancer.Oncotype DX, a very expensive gene test, is routinely used in hospitalsto predict chemotherapy benefit and recurrence risk of patients withbreast, colon and prostate cancer. The present TMEM219 assay is anexcellent tool for replacing or complementing the Oncotype DX test.

FIG. 4 depicts a schematic overview of the mechanism of action of theIGFBP-3/TMEM219 axis in cancer.

Example 2. Agonist Antibodies and Cancer

The naturally occurring TMEM219 agonist does not in and of itselfrepresent a useful therapeutic agent due to significantpost-translational modification: proteolysis induced by tumor activatedproteases attenuates TMEM219 antitumor signaling. Thus, monoclonalantibodies (mAbs) that activate TMEM219, i.e. “agonist antibodies” werecreated. The process of manufacturing mAbs has been standardized in theart and mAbs are known to exhibit robust stability within the body,without inducing major deleterious side effects. The “agonist mAb”approach advantageously precludes the need to use toxic compounds tokill the cancer cells.

A panel of TMEM219 specific monoclonal antibodies was generated and twoexemplary antibodies were sequenced (#245 and #274, FIGS. 5A and B).Further, a TMEM219 #274-human IgG1 chimera which acts like the TMEM219natural agonist was also developed (FIG. 5C). As shown in FIG. 6A-C,treatment with one of the hybridoma cell-produced TMEM219 agonist mAbs[TMEM219 mAb #2 (#274)] significantly inhibited not only MCF-7estrogen-responsive breast cancer cell growth but also MDA-MB231 TNBCcell growth. However, MCF-10A normal immortalized mammary epithelialcell growth was not inhibited despite expressing a similar level ofTMEM219 at the mRNA and protein levels. It was further observed thatTMEM219 mAb #2 (#274) inhibits the growth of both breast cancer celllines in a dose dependent manner with 70% growth inhibition at theconcentration of 30 nM (p<0.001).

As described below, TMEM219 agonist mAbs have tumor suppressive activitynot only on cancer cells in culture, but also in animal models of humancancer (models representing non-small cell lung cancer, triple-negativebreast cancer (TNBC), colon cancer, and prostate cancer). In addition,TMEM219 agonist mAbs also suppress the tumor-activated signalingcritical to tumor angiogenesis, metastasis and radio-/chemo-resistance.Of high importance is the fact that, despite strong anti-cancer cellactivity, the TMEM219 agonist antibodies have no deleterious cellkilling effect on normal, non-tumor cells.

Breast Cancer

The antitumor and anti-metastatic effects of TMEM219 agonist mAbs wasinvestigated using a bioluminescent orthotopic MDA-MB231 triple negativebreast cancer (TNBC) mouse model. In vitro data using MDA-MB231 cellsexpressing dtTomato-Luciferase clearly demonstrates that treatment ofTMEM219 agonist mAb #2 (#274) resulted in induction of caspase-dependentapoptosis as shown significant activation of caspase-3 and subsequentdecrease of total PARP, and suppression of tumor-activated NF-kappa Bsignaling, as shown by the decrease of phospho- and total p65 NF-kappa B(FIG. 7A). Treatment of 30 nM agonist mAb #2 (#274) for 4 days resultedin significant cell death in bioluminescent MDA-MB231 cells (FIG. 7B).As similarly observed with TMEM219 #274 mAb, TMEM219 #274-hIgG1 chimeradetects TMEM219 in MCF-7 cell lysates (FIG. 7C, left panel) and alsoactivates caspase-3 and inhibits tumor-activated NF-kappaB signaling inbioluminescent MDA-MB-231 cells (FIG. 7C, right panel). Furthermore,TMEM219 knockdown using CRISPR/Cas9 gene editing techniques resulted incomplete abolishment of TMEM219 agonist mAb-induced cell growthinhibition (FIG. 7D). When two different sequence targeted single guideRNA (sgRNA) constructs were transfected into bioluminescent MDA-MB231cells, TMEM219 expression was suppressed up to 95% by each individualconstruct (upper panel sgRNA-1 and sgRNA-2). Treatment with 100 nMTMEM219 agonist mAbs resulted in significant growth inhibition in thecontrol (68% inhibition), whereas only 10% and 6% growth inhibition wasobserved in sgRNA-1 and sgRNA-2-transfected cells, respectively, afterTMEM219 agonist mAbs treatment (bottom panel). These data demonstratethat TMEM219 agonist mAbs-induced anticancer effects are mediatedthrough TMEM219 antitumor signaling in human cancers (FIG. 7D).

The antitumor and anti-metastatic effects of TMEM219 agonist mAbs wereinvestigated using a bioluminescent orthotopic breast tumor mouse model.MDA-MB231 cells expressing dtTomato-Luciferase were injected into thefourth mammary fat pad of 8 week old NOD-SCID-IL2γR−/− mice. As shown inFIG. 8A-C, administration of a low dose of TMEM219 mAb (1 mg/kg bodyweight) resulted in tumor shrinkage up to 25% (p<0.01) at day 29 aftertumor cell injection. No detectable adverse effects, determined e.g. bybody weight change and macroscopic damages in major organs such asliver, kidney and spleen, were observed.

The expression profiles of TMEM219 and the antitumor effect of TMEM219agonist mAb were examined in Patient Derived Xenograft (PDX) TNBC cells.As shown in FIG. 9, TMEM219 is readily detectable in all TNBC PDX cellstested at the protein and mRNA levels (FIGS. 9A and 9B). Thoseexpression levels were comparable to the established TNBC cells,MDA-MB231 and MDA-MB468. In addition, immunohistochemistry data clearlydemonstrate that TMEM219 is expressed in both PDX tumors and presentmainly in cell membrane and cytoplasmic region but not in the nucleus(FIG. 9C). Furthermore, TMEM219 agonist mAb #2 (#274) treatment resultedin a significant growth inhibition not only in chemodrug-sensitiveWHIM30 but also in chemodrug-resistant WHIM2 PDX TNBC cells (FIG. 9D).

In addition, bioluminescent orthotopic WHIM30 PDX mice showed thatadministration of a low dose of TMEM219 agonist mAb #274 (1 mg/kg bodyweight) resulted in tumor shrinkage of up to 29% at day 30 after tumorcell injection (FIG. 10A). No apparent body weight or damage in majororgans was observed in TMEM219 agonist mAb administrated mice (FIG.10B). Consistent with tumor volume data, the size and weight of tumorsisolated from TMEM219 agonist mAb administrated mice was significantlyreduced compared with those from mouse IgG administrated mice (FIG.10C). The 25% reduction of tumor weight observed in mAb administeredmice was comparable to the 29% tumor shrinkage shown in FIG. 8A. It isnoted that these promising antitumor data were obtained from singleminimal dose. These results clearly indicate that the TMEM219 anti-tumorsignaling pathway is still functional in TNBC and that TMEM219 mAbsrepresent a new therapeutic intervention for high mortality TNBC.

Colon Cancer (CAC)

An in vivo study was conducted using the Dextran SulfateSodium-Azoxymethane (DSS-AOM) mouse model, which is well accepted as aCAC animal model. The results clearly demonstrated that a neutrophilprotease inhibitor, α1-antitrypsin (AAT) treatment during late-stage CACresulted in a significant reduction in the frequency and size of tumorsin mice harboring established CAC (FIG. 13A-E). Furtherimmunohistochemistry (IHC) data clearly demonstrated significantsuppression of intramucosal adenocarcinoma (TIS) formation as well asIL-6, a cell proliferation marker PCNA and a major component ofneutrophil azurophilic granules myeloperoxidase (MPO) in colon tissue.In addition, AAT treatment results in a significant increase of TMEM219agonist in colon tissue as well as in circulation. These data stronglysuggests that antitumor effect of AAT may be attributed to reducedTMEM219 natural agonist proteolysis, thereby enhancing TMEM219 naturalagonist/TMEM219-mediated antitumor/anti-inflammatory, and furtherameliorating neutrophil-activated cytokine function such as activationof the IL-1β/IL-6 axis in CAC.

Therapeutic potential of TMEM219 agonist mAbs was further tested forcolorectal cancer using an AOM/DSS CAC mouse model. The in vivopreclinical data clearly demonstrate that mice treated with TMEM219agonist mAb #2 (#274) showed a dramatic suppression of tumor number andsize (FIG. 14A). Similarly seen with TMEM219 natural agonist treatment,treatment with TMEM219 agonist mAbs inhibits HT-29 colon cancer cellgrowth (FIG. 14B) and tumor-activated NF-kappa B signaling as showndecrease of phosphorylated-NF-kappa B and -I kappa B alpha (FIG. 14C).

Lung Cancer

Smoking contributes to 80 percent and 90 percent of lung cancer deathsin women and men, respectively. To date, much evidence exists confirmingthe involvement of tobacco carcinogens such as NNK in lungtumorigenesis. It is known that, NNK, the most potent tobaccocarcinogen, enhances cell proliferation of BEAS-2B, normal lungepithelial cells, and concomitantly suppresses TMEM219 natural agonistbut not TMEM219 expression through DNA methylation. Decreased TMEM219natural agonist expression, and elevated levels of phospho-Akt,phospho-p65-NF-kappaB, and cyclin D1 were detected in tobaccocarcinogen-induced tumorigenic derivatives of BEAS-2B. Overexpression ofTMEM219 natural agonist in NNKA, one of the derivatives, suppressedNF-kappaB activity and induced apoptosis whereas suppression of TMEM219with its specific shRNA hindered TMEM219 natural agonist-inducedsuppression of NF-κB and induction of cell death. These obsevationsindicate that the observed anti-tumor actions of TMEM219 natural agonistis mainly mediated via TMEM219 in NNKA cells. Taken together, thisunique tumor specific antiproliferative and proapoptotic property ofIGFBP-3/TMEM219 axis provides a strong evidence for its therapeuticvalue for lung cancer. However, IGFBP-3 itself does not constitute anexcellent targeted therapy for lung cancer due to its significantdegradation by tumor-induced proteases such as MMPs and ADAM28, therebyattenuating IGFBP-3's antitumor function (FIG. 11). Two TMEM219 mAbs#245 and #274 possess antitumor effects in lung cancer cells but not innormal lung epithelial cells (FIG. 12). In addition, these findingsdemonstrate that TMEM219 is a key antitumor signaling and a therapeutictarget in NSCLC.

Example 3

According to World Health Organization estimates, overweight and obesitynow overshadow underweight and malnutrition as significant causes ofpremature death. Nearly two-thirds of adults in the United States areoverweight or obese, and obesity is a major risk factor for a myriad ofserious comorbidities including hypertension, type 2 diabetes mellitus(T2DM), cardiovascular disease (CVD), and other metabolic disorders.Additionally, rapidly increasing rates of obesity in children and youngadults has been observed and is resulting in immediate and lifelongmetabolic disease risk. Lifestyle changes to counteract obesity andphysical inactivity have been emphasized as the first line of defenseagainst progression to T2DM, however there has been no significantdecrease in the incidence of obesity. More effective preventive andtherapeutic strategies are needed to thwart obesity and associatedmetabolic complications.

Insulin resistance (IR) represents a common metabolic derangement thatcontributes to the development of many obesity-related comorbiditiesincluding T2DM. Although it is generally established that low-gradeadipose tissue inflammation contributes substantially to the burden ofIR, the pathophysiology underlying the development of IR is complex andmultifactorial. Thus, a clearer understanding of the mechanisms leadingto obesity-associated IR is necessary to identify novel targets for theprevention and treatment of many IR-driven conditions such as T2DM. Theendocrine paradigm suggests that visceral fat in obesity, consistingprimarily of adipocytes, secretes various pro-inflammatory adipokinessuch as tumor necrosis factor (TNF), leptin, visfatin, resistin, andinterleukin (IL)-6 creating a state of local thus accelerating eventsleading to systemic IR, T2DM and metabolic syndrome. Recent studies havefurther identified that obesity-induced inflammatoryadipokines/cytokines interfere with insulin signaling in visceraladipocytes by decreasing the levels of insulin receptor substrate-1(IRS-1), glucose transporter-4 (GLUT4) and adiponectin leading to astate of IR via autocrine/paracrine influences.

IGFBP-3 inhibits TNF-alpha-induced NF-kappa B activity through IGFBP-3R,thereby restoring insulin signaling and negating TNF-alpha-inducedinhibition of glucose uptake in human primary adipocytes, suggestingthat the IGFBP-3/IGFBP-3R system plays an important role incytokine/adipokine-induced IR in visceral adipocytes. Furthermore, thereis a decrease in functional intact IGFBP-3 levels and an increase inIGFBP-3 degradation (proteolysis) in the circulation of overweight andobese adolescents when compared with their non-obese counterpart.Moreover, a significant inverse correlation is observed betweenfunctional IGFBP-3 and adiposity parameters such as waist circumference,body mass index and homeostasis model assessment of insulin resistance(HOMA-IR). These findings suggest that inflammation-induced decrease ofintact IGFBP-3 due to decreased IGFBP-3 production as well as increasedIGFBP-3 degradation (proteolysis) in overweight and obese populationlikely results in reduced levels of functional IGFBP-3 in circulation,effectively blunting the anti-inflammatory and insulin-sensitizingfunctions of the IGFBP-3/IGFBP-3R system in adipose tissue. It furthersuggests a regulatory role for the IGFBP-3/IGFBP-3R system in glucosehomeostasis.

An in vivo study using Aralast (a clinical formulation of humanAlpha1-Proteinase Inhibitor, “AAT”) using a diet-induced obese (DIO)mouse model showed that mice fed with high fat diet (HFD) for 15 weeksresulted in a significant insulin resistance (IR) shown by ITT whereasAAT treated HFD mice showed a significant improvement in IR (FIG. 15A).In addition, H&E staining of liver and visceral fat tissues demonstratedthat AAT reduced hepatic steatosis and visceral fat inflammation (FIG.15B). Furthermore, a significant decrease of serum IGFBP-3 proteolysiswas observed in AAT administrated HFD mice. (FIG. 15C). These in vivodata clearly indicates therapeutic potential of AAT and TMEM219 agonists(IGFBP-3 and TMEM219 agonist antibodies).

In order to identify the functional significance and therapeuticpotential of IGFBP-3R agonistic antibodies in IGFBP-3-inducedanti-inflammatory and insulin sensitizing effects studies were conductedin which IGFBP-3R agonist mAbs #245 and #274 and non-agonistic IGFBP-3Rmonoclonal antibodies (#C314) were employed in the presence of insulinand TNF-alpha in fully differentiated adipocytes. As shown in FIG. 16,IGFBP-3R agonist mAbs #245 and #274, but not non-agonistic mAb, restoredTNF-alpha-induced inhibition of glucose uptake in primary adipocytes.These results show that IGFBP-3R agonist mAbs inhibit TNF-α-inducedinsulin resistance by inhibiting TNF-alpha-induced NF-kappa B activityin adipocytes.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

1. An antibody that binds to and activates the insulin-like growthfactor-binding protein 3 receptor (IGFBP-3R), wherein the antibodycontains the complementarity determining regions (CDRs) SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, and theamino acid sequence ATS.
 2. The antibody of claim 1, wherein theantibody comprises a detectable label.
 3. A method of treating anIGFBP-3R expressing cancer in a patient in need thereof, comprising:administering to said patient a therapeutically effective amount of anantibody that binds to and activates the insulin-like growthfactor-binding protein 3 receptor (IGFBP-3R), wherein the antibodycontains the CDRs SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ IDNO: 21, SEQ ID NO: 23, and the amino acid sequence ATS.
 4. The method ofclaim 3, wherein said IGFBP-3R expressing cancer is breast cancer, coloncancer, lung cancer, ovarian cancer, pancreatic cancer, liver cancer,head and neck cancer, prostate cancer or a liquid tumor.
 5. The methodof claim 4, wherein the liquid tumor is a leukemia.